Three Dimensionally and Randomly Oriented Fibrous Structures

ABSTRACT

A randomly-oriented 3-D fibrous structure and a method for making the same. The method involves electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and a surfactant; to form one or more fibers that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a non-provisional application claiming thebenefit of U.S. Provisional Patent Application 61/668,269, filed Jul. 5,2012, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under a grant from theU.S. Dept. of Agriculture (NEB 37-037). The government has certainrights to this invention.

FIELD OF THE INVENTION

This invention is directed to electrospinning three-dimensionally (3D)and randomly oriented fibrous structures from various polymer sources,including plant proteins, animal proteins, and synthetic polymers.

BACKGROUND OF INVENTION

Ideal tissue engineering scaffolds should be capable of closelymimicking the topographies and spatial structures of nativeextracellular matrices (ECMs) to facilitate cells to grow anddifferentiate following the patterns similar to that found in nativetissues and organs. Morphologies of ECMs vary according to functions oftarget tissues and cell types in the tissues. For example, in skintissue, the top layer is formed by compact packing of epithelial cellson a two-dimensional (2D) fibrous ECM basement membrane.Three-dimensional spatial spreading of fibroblasts and immune cellsoccurs in the interior region of the skin tissue, and correspondinglythe ECMs are constructed by stereoscopically and randomly orientedultrafine protein fibers. Fibrous structures with 3D orientation andrandom distribution can also be found in native ECMs in breast, liver,bladder, lung, and many other organs and tissues. It has been reportedthat cells cultured on flat 2D substrates may differ considerably inmorphology and differentiation pattern from those cultured in morephysiological 3D environments. Therefore, it is reasonable to fabricatescaffolds with particular morphologies and structures according tocategories and functions of original native tissues.

Due to its simplicity and high efficiency, electrospinning has beenwidely employed to fabricate tissue engineering scaffolds composed ofnano- or submicrometer-fibers from numerous materials. However,conventional electrospun structures typically form 2D scaffolds withfibers aligned parallel to the collector and cells cultured on theconventional electrospun scaffolds could only develop into flat shapes.The functions and differentiation of many flattened cells could notresemble the native stereoscopic cells. Furthermore, small pore sizes,owing to the close arrangement of fibers, restricted access of cells tothe interior of conventional electrospun scaffolds. Thus, onconventional electrospun scaffolds, cells could mainly spread anddistribute within a shallow depth beneath the surface.

To date, many 3D electrospinning techniques have been developed tofabricate electrospun scaffolds with larger pores and higher porosity toimprove cell accessibility of the scaffolds. Examples of such techniquesinclude wet electrospinning, electrospinning with integration of coarsefibers, and electrospinning with porogens (e.g., dry ice, salt, orsucrose), which are based on the concept of including a “blocking agent”to increase the distances between electrospun fibers in order to lead todeeper penetration of cells into interior of scaffolds. Nonetheless,these techniques failed to change the planar orientations of theelectrospun fibers and as a result the scaffolds tended to have parallelfibrous layer-by-layer structures. Additionally, the parallel fibrouslayer-by-layer structures tended to not have pores that extend very farin the thickness direction as compared to in the planar directions. As aresult, there was limited improvement in scaffold porosity and cellscultured thereon tended to have flattened morphologies rather thanstereoscopically developed cells in many native tissues. Still further,there have been attempts to fabricate 3D electrospun scaffolds based onthe electrostatic repulsion between as-spun fibers. Despite their fluffyappearances, such scaffolds still had a layer-by-layer structure ofplanar mats and parallel oriented fibers.

In view of the foregoing, a need still exists for randomly-oriented 3-Dfibrous structures and a method for making the same via electrospinning.

SUMMARY OF INVENTION

In one embodiment, the present invention is directed to a method ofmaking a randomly-oriented 3-D fibrous structure. The method comprisingelectrospinning a spinning dope with an electrospinning apparatus,wherein the spinning dope comprises: a solvent; a polymer dissolved inthe solvent, wherein the dissolved polymer is in subunits havingmolecular weights that are about 5 to about 150 kDa; and a surfactant;to form one or more fibers that comprise a polymer-surfactant complexand that arrange randomly and evenly in three dimensions when contactinga collecting board of the electrospinning apparatus thereby forming therandomly-oriented 3-D fibrous structure.

In one embodiment, the present invention is directed to a method ofmaking a randomly-oriented 3-D fibrous structure. The method comprisingelectrospinning a spinning dope with an electrospinning apparatus,wherein the spinning dope comprises: a solvent; a polymer dissolved inthe solvent, wherein the dissolved polymer is in subunits havingmolecular weights that are about 5 to about 150 kDa, and wherein thepolymer is selected from the group consisting of protein, syntheticpolymer, and combinations thereof; and an anionic surfactant at about 5to about 300 percent by weight of the polymer; to form one or morefibers of a fineness that is about 50 nm to about 100 μm and thatcomprise a polymer-surfactant complex and that arrange randomly andevenly in three dimensions when contacting a collecting board of theelectrospinning apparatus thereby forming the randomly-oriented 3-Dfibrous structure that further comprises interconnected pores havingsizes that are about 10 to 2000 μm and that has a porosity that is about60 to about 99.9% by volume.

In one embodiment, the present invention is directed to arandomly-oriented 3-D fibrous structure comprising: one or more fibersthat comprise a polymer-surfactant complex, wherein the fiber(s) havelengths that are at least about 100 nm and finenesses that are about 50nm to about 100 μm, and are arranged randomly and evenly in threedimensions throughout the randomly-oriented 3-D fibrous structure; andinterconnected pores having sizes that are about 10 to 2,000 μm, whereinthe pores comprise about 60 to about 99.9% by volume of therandomly-oriented 3-D fibrous structure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 contains schematic diagrams of fibers being deposited viaelectrospinning in which the 2D column contains images (a), (b), and (c)showing the parallel deposition of conventional electrospun fibers andthe 3D column contains images (d), (e), and (f) showing the depositionof fibers according to the present invention.

FIG. 2 contains photographic images of fibers being deposited viaelectrospinning in which the (a) column contains images 1, 2, and 3,which were taken at 0.125 second intervals showing the deposition ofconventional, parallel-aligned electrospun fibers and the (b) columncontains images 1, 2, and 3, which were taken at 0.125 second intervalsshowing the vertical deposition of fibers according to the presentinvention.

FIG. 3 is a photographic comparison of a zein electrospunrandomly-oriented 3-D fibrous structure and a zein electrospun 2Dfibrous structure; the structures are of the same weight.

FIG. 4 are images of a zein electrospun randomly-oriented 3-D fibrousstructure: (a) is a SEM 70x top view image; (b) is a SEM 70x side viewimage; (c) is a CLSM 100x 45 degree front view image; and (d) is a CLSM100x side view image.

FIG. 5 are CLSM 60x images of sequential sections of 2D [(a) 1, 2, 3,and 4] and randomly-oriented 3-D fibrous structures [(b) 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, and 12] each taken at increasing 10 μm depths inwhich the lighter network is F-actin in NIH 3T3 cells stained withPhalloidin 633 after being cultured in the structures for 72 hours.

FIG. 6 is graph of MTS assay results of attachment (4 hours) andproliferation (24, 72, 120, and 168 hours) of NIH 3T3 fibroblase cellson 2D and 3D zein electrospun fibrous structures.

FIG. 7 are CLSM images showing the proliferation of NIH 3T3 cells on 2Dand 3D zein electrospun fibrous structures about 48 hours after seeding(stained with Phalloidin 633 and Hoechst 33342). The three-dimensionalreconstruction of the 2D fibrous structure is (a): x-y projection (I);x-z projection (II′); and y-z projection (III). The three-dimensionalreconstruction of the 3D fibrous structures is (b): x-y projection (I′);x-z projection (II′); and y-z projection (III′).

FIG. 8 is a photographic comparison of an electrospun randomly-oriented3-D PEG fibrous structure prepared with SDS and an electrospun 2Dfibrous structure; the structures are of the same weight.

FIG. 9 is a photograph of a PEG electrospun randomly-oriented 3-Dfibrous structure prepared with a nonionic surfactant.

FIG. 10 is a SDS-PAGE of soy protein, and wheat glutenin, wherein lane 1shows standard protein markers, lane 2 is untreated soy protein, lane 3is cysteine extracted soy protein, lane 4 is NaOH extracted soy protein,lane 5 is untreated wheat glutenin, and lane 6 is cysteine extractedwheat glutenin.

FIG. 11 is a photograph of a soy protein electrospun randomly-oriented3-D fibrous structure.

FIG. 12 is are photographs of soy protein electrospun randomly-oriented3-D fibrous structure, wherein (a) is before immersion in PBS and (b) is3 days after immersion in PBS; scale bar is 20 μm.

FIG. 13 is a photograph of a feather keratin electrospunrandomly-oriented 3-D fibrous structure.

FIG. 14 is a photograph of a wheat glutenin electrospunrandomly-oriented 3-D fibrous structure.

FIG. 15 is a photograph of a wheat gliadin electrospun randomly-oriented3-D fibrous structure.

FIG. 16 is a photograph of a casein electrospun randomly-oriented 3-Dfibrous structure.

FIG. 17 is photograph of a peanut electrospun randomly-oriented 3-Dfibrous structure.

FIG. 18 (a) is graph showing the relationship between specific porevolume of electrospun scaffolds and surface resistivity (the dashed lineshows the simulated relation using power function) and FIG. 18 (b) is agraph showing the effect of sodium dodecyl sulfate (SDS) and NaCl onsurface resistivity of PEG films based on the proportions of SDS topolymer (the molar concentration of NaCl was the same as that of SDS ateach point).

FIG. 19 (a) are photographs of the deposition process of zein onto aninsulator covered collector at time intervals of 0.125 seconds betweeneach image (the spinning dope consisted of 25 wt % zein and 25 wt %SDS); FIG. 19 (b) is a photograph of the as-spun zein scaffold; and FIG.19( c) is a CLSM 45° image at magnification of 100× of the above as-spunzein scaffold.

DETAILED DESCRIPTION OF INVENTION

An embodiment of the present invention is directed to a method of makinga randomly-oriented 3-D fibrous structure. The method comprisingelectrospinning a spinning dope with an electrospinning apparatus,wherein the spinning dope comprises: a solvent; a polymer dissolved inthe solvent, wherein the dissolved polymer is in subunits havingmolecular weights that are about 5 to about 150 kDa; and a surfactant;to form one or more fibers that comprise a polymer-surfactant complexand that arrange randomly and evenly in three dimensions when contactinga collecting board of the electrospinning apparatus thereby forming therandomly-oriented 3-D fibrous structure. Typically, the electrospinningis conducted at a temperature that is about 25 to about 70° C.

As used herein with respect to the present invention, the“randomly-oriented 3-D fibrous structure” is intended to mean fibrousstructure in which the one or more fibers thereof are randomly orientedin all three dimensions rather than only being randomly oriented in twodimensions as can be found in conventional layer-by-layer, planarstructures. The degree of random orientation of a fibrous structure maybe quantified by, for example, identifying a cubic centimeter volume ofsaid fibrous structure and identifying any particular imaginary plane ofany orientation within said volume, identifying the number of fibersintersected or fiber intersections both of which are “intersections”with said plane, and identifying the number of said intersections inwhich said intersected fiber(s) are at angles greater than 30 degreesrelative to said plane. In certain embodiments of the present invention,at least ½, ⅝, ⅔, and ¾ of the intersected fibers are at angles greaterthan 30 degrees with the plane.

As used herein the terms “scaffold” and “fibrous structure” are intendedto have the same meaning and may be used interchangeably.

Mechanism

Without being bound to any particular theory, it is believed that themechanisms of 2D electrospinning and an embodiment of 3D electrospinning(of the present invention produce) are depicted in FIG. 1. In bothconventional and 3D electrospinning, at the beginning, the liquiddroplet acquired negative charges and then was elongated into fibers.For conventional electrospinning, relatively few of the electrons aretransferred to the collector at the moment the fiber ends hit thecollector, owing to high surface resistivity of the fibers. The fiberswith a relatively large amount of remaining electrons are stronglyattracted to the positively charged collector. As a consequence,conventional 2D electrospun scaffolds have fibers oriented parallel tothe collecting board and the fibrous structures are relatively tightlypacked.

In contrast, the depicted 3D electrospinning embodiment is believed tobe based on repulsive electrical force between fibers and the collector.More specifically, the method of the present invention, involvesincluding a surfactant to decrease the surface resistivity of a fiber tothereby increase the transfer of charge from the fiber surface to thecollector. When the fiber(s) strike the collector, surface staticelectricity transfers to the board in a faster manner, which results inless negative static electricity remaining on the fiber(s), anddecreased attraction between fiber(s) and the collector. In some cases,the near portion of the fibers may even carry positive charges and maybe repulsed by the collector, while the farther end of the fiber(s) isstill attracted and moves towards the board. As a result, fiber(s) arecollected onto the board in multiple orientations to form loose andfluffy 3D scaffolds with randomly-oriented fiber(s).

It is believed that the above-described mechanisms have been shown byexperimental results shown in FIG. 2 in which FIG. 2 a is conventionalelectrospinning and FIG. 2 b is electrospinning in accordance with thepresent invention at 0.125 second intervals between sequentialphotographs with the the white objects PEG fibrous bulks.

Polymer

As indicated above, the spinning dope comprises a polymer, which may beany appropriate polymer such as protein, synthetic polymer, andcombinations thereof. Proteins may be of particular interest becausethey are preferred in biomedical applications due to their molecularsimilarity to native ECMs, and tend to be widely available at low cost.For example, plant proteins, zein and soy protein, and animal proteins,such as keratin and collagen, have been proved to be supportive to cellgrowth in in vitro and in vivo studies. In addition, plant and animalproteins are widely available at a low cost and they are considered tobe a renewable resource. The method of the present invention may beconducted using one or more proteins selected from the group consistingof plant protein, animal protein, and combinations thereof.

Many such proteins, however, are considered to be highly-linked and as aresult tend to have limited solubility in water and organic or alcoholicsolvents. In general, proteins with cysteine content higher than 1% inits amino acid composition are considered to be highly-crosslinkedproteins. Exemplary highly-crosslinked proteins, including keratin,which has a cysteine content of about 7%, soy protein has a cysteinecontent of about 1.3%, and wheat glutenin a cysteine content of about2%. In particular, electrospinning highly crosslinked proteins, such assoy protein, feather keratin and wheat glutenin, into fibrous structuresvia electrospinning has not been possible due to their insolubility invarious solvents.

With reductant and denaturant in the spinning dope, coarse fibers fromsoy protein and wheat gluten via wet spinning have been produced. Reddy,N. and Y. Yang, Novel Protein Fibers from Wheat Gluten,Biomacromolecules, 2007, 8(2), p. 638-643; Reddy, N. and Y. Yang, Soyprotein fibers with high strength and water stability for potentialmedical applications, Biotechnology Progress, 2009, 25(6), p. 1796-1802.However, electrospinning with pure protein required much betterdissolution of proteins, and thus the spinning dope for wet spinningfailed to be electrospun. Highly hydrolyzed soy protein with smallmolecules had been electrospun with PEG, which accounted for thespinability. Vega-Lugo, A. C. and L. Loong-Tak, Electrospinning of SoyProtein Isolate Nanofibers, Journal of Biobased Materials and Bioenergy,2008, 2(3), p. 223-230. Hydrolyzed soy protein in its pure form,however, had not been electrospun.

The present invention of electrospinning may be practiced, however, withhighly crosslinked protein by dissolving the protein in manner thatpreserves the protein subunits with appropriate molecular weightsranging from 5 to 150 kDa. Other methods of dissolving highlycrosslinked proteins (e.g., U.S. Pat. Pub. No. 2006/0282958, Yang etal., entitled Process for the Production of High Quality Fibers fromWheat Proteins and Products Made From Wheat Proteins) allowed for thespinning of coarse fibers but were not suitable for electrospinning ofrelatively fine fibers. The electrospinning method of the presentinvention may be practiced with highly crosslinked proteins by using areducing agent such a thiol, a sulfite, or a sulfide to break thedisulfide bonds of the highly crosslinked proteins. For example,cysteine, an environmentally-benign thiol, may be used as a reducingagent to break the disulfide bonds and achieve dissolution ofhighly-crosslinked proteins. The amount of the reducing agent in thesolution may be varied from about 1% to about 50% based on the weight ofproteins. The solvent for the protein and cysteine was a 4-8 M ureasolution with pH of 8 to 12, adjusted using 50 wt % sodium hydroxidesolution. The weight ratio of protein-containing materials to ureasolution may be varied within a range from, for example, 1:5 to 1:30.The temperature for the dissolution may be within a range from about 20°C. to about 90° C. Examples of additional appropriate thiols includemethanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, butanethiol,tert-butyl mercaptan, pentanethiols, thiophenol, thioacetic acid,coenzyme-A, glutathione, 2-mercaptoethanol, dithiothreitol,2-mercaptoindole, 3-mercaptopropane-1,2-diol. Examples of appropriatesulfites and sulfides include sodium sulfite potassium sulfite, sodiumbisulfite, potassium bisulfite, sodium sulfide, potassium sulfide,sodium metabisulfite, and potassium metabisulfite. The conditions forthe reactions include the thiol/sulfite/sulfide at concentration(s) ofabout 0.5% to about 50% based on the weight of proteins, a pH from 3 to12 for a duration of about 30 minutes to about 24 hours at a temperatureof about 20° C. to about 90° C.

In view of the foregoing, the method of the present invention may, incertain embodiments, be conducted using appropriately dissolvedhighly-crosslinked plant protein, highly-crosslinked animal protein, andcombinations thereof. When using proteins, including highly-crosslinkedproteins, experimental results to date have shown the aging thedissolved protein at an aging temperature that is about 20° C. to about90° C. for an aging duration that is about 0.5 to about 48 hours beforeconducting the electrospinning may be advantageous. For example, it hasbeen found that such aging may provide a higher degree ofdisentanglement of the protein molecules to improve the spinnability ofthe protein spinning dope.

Exemplary plant proteins include wheat gluten, wheat gliadin, wheatglutenin, soy protein, camelina protein, peanut protein, canola protein,sorghum protein, rice protein, millet protein, sunflower seed protein,pumpkin seed protein, mung bean protein, red bean protein, chickpeaprotein, green pea protein, and combinations thereof;

Exemplary animal proteins include chicken feather, egg white, woolkeratin, casein, silk, fibrin, collagen, gelatin, hair keratin, hornkeratin, nail keratin, whey protein, and combinations thereof.

Exemplary synthetic polymers include polyethylene glycol (PEG), polylactic acid (PLA), poly glycolic acid (PGA), polyhydroxyalkanoates(PHAs), poly(lactic-co-glycolic acid) (PLGA), poly-3-hydroxybutyrate(PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH),poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and combinationsthereof.

As indicated above, the solution may comprise one, two, or even morepolymers. In certain embodiments with more than a single polymer, thepolymer that is in the largest amount (usually more than half of thetotal polymer content) is often referred to as the “primary” polymer,component, or material and the additional polymers are often referred toas “secondary” polymer(s), component(s), or material(s) withconcentrations that are from about 0.5% to about 50% by weight of theprimary polymer.

Surfactant

As indicated above, the spinning dope comprises a surfactant, which maybe any appropriate surfactant. The hydrophobic portions of surfactantbond with polymers through hydrophobic interaction to form aprotein-surfactant complex. Without being bound to a particular theory,it is believed that the hydrophilic portions, including functionalgroups that carry positive or negative charges and polar unchargedgroups, gather on the fiber surface and thus effectively increase thesurface conductivity (or lower the surface resistivity) by introducingsurface water layer, which facilitates delivery of charges on thesurface.

The surfactant(s) may be of different hydrocarbon chain lengths anddifferent electrical properties, including anionic surfactants, cationicsurfactants, nonionic surfactants, zwitterionic surfactants. In anembodiment, combinations of surfactants may be used, as appropriate. Inanother embodiment, the surfactant is one or more anionic surfactants.

Typically, the spinning dope has a concentration of the surfactant thatis about 5 to about 300 percent by weight of the polymer. In anotherembodiment, the spinning dope has a concentration of the surfactant thatis about 50 to about 150 percent by weight of the polymer.

Exemplary anionic surfactants include sodium dodecyl sulfate, sodiumdodecyl benzenesulfonate, sodium lauryl sarcosinate,perflourobutanesulfonic acid, ammonium lauryl sulfate, sodium stearate,sodium pareth sulfate, dioctyl sodium sulfosuccinate, potassium laurylsulfate, sodium laureth sulfate, sodium myreth sulfate, and combinationsthereof.

Exemplary cationic surfactants include benzalkonium chloride,cetrimonium bromide, tetramethylammonium hydroxide, octenidinedihydrochloride cetyl trimethylammonium bromide, hexadecyl trimethylammonium bromide, cetyl trimethylammonium chloride (CTAC),cetylpyridinium chloride (CPC), benzalkonium chloride (BAC),benzethonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane,dimethyldioctadecylammonium chloride, cetrimonium bromide,dioctadecyldimethylammonium bromide (DODAB), and combinations thereof.

Exemplary nonionic surfactants include decyl glucoside, octyl phenolethoxylated, polysorbate 80, polysorbate 20, and combinations thereof.

Exemplary zwitterionic surfactants include cocamidopropylhydroxysultaine, cocamidopropyl betaine, lecithin,3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), and combinations thereof.

Solvent

As indicated above, the spinning dope comprises a solvent, which may beany appropriate solvent. Exemplary solvents include water, phosphatebuffered saline (PBS), carbonate buffer, tris-glycine buffer, boratebuffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid (CHES)buffer, citric buffer, ethanol, chloroform, 1,4-dioxane, methanol,ethylene glycol, acetone, ethyl acetate, methyl acetate, hexane, petrolether, citrus terpenes, diethyl ether, dichloromethane,dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO),formic acid, n-butanol, isopropanol (IPA), n-propanol, acetic acid,nitromethane, dichloromethane, and combinations thereof. In anotherembodiment, the solvent is selected from the group consisting of water,phosphate buffered saline (PBS), carbonate buffer, tris-glycine buffer,borate buffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid(CHES) buffer, citric buffer, chloroform, and combinations thereof.

Additional Spinning Dope Components and Method Steps

In addition to the above-described components, the spinning dope maycomprise one or more additional components such as a cross-linker, asacrificial component, or a combination thereof. Alternatively or inaddition to including such additional components in the spinning dope,the method may further comprise contacting the fibers with a secondsolution that comprises a second cross-linker, a surface decorator, or acombination thereof.

Cross-linker(s)

Exemplary cross-linkers include polycarboxylic acid which contains atleast three carboxylic acid groups (such as citric acid, iso-citricacid, propane-1,2,3-tricarboxylic acid, trimesic acid, aconitic acid,mellitic acid, 1,2,3,4-Butane tetracarboxylic acid (BTCA)), oxysucrose,genepin, glutaraldehyde, oxaldehyde, NHS esters, maleimides,carbodiimide, isocyanate, and combinations thereof. If present in thespinning dope and/or in a cross-linking solution, the concentration ofcross-linker is typically about 0.01 mol/L to about 10 mol/L. In anembodiment in which the spinning dope comprises a first cross-linker andthe method further comprises contacting the fibers with a cross-linkingsolution that comprises a second cross-linker, the first and secondcross-linkers may be independently selected and as such may be identicalor different (e.g., in terms of material, concentration, or both). In anembodiment of the present invention, the first and second cross-linkersare independently selected from the group consisting citric acid,1,2,3,4-Butane tetracarboxylic acid (BTCA), oxysucrose, genepin,glutaraldehyde, oxaldehyde, and combinations thereof.

Sacrificial Component

Exemplary sacrificial components include PEG, egg white protein, zein,wheat gliadin, and other materials that may be dissolved with theabove-described polymer(s) before electrospinning at concentrations ofabout 0.5% to about 50% based on the weight of the polymer. Thesacrificial component(s) may be removed from the spun fibrous structurevia rinsing with water, organic solvents or alcoholic solvents.

Surface Decorator

Exemplary surface decorators of fibers include peptides such asArg-Gly-Asp (RGD), Ile-Lys-Val-Ala-Val (IKVAV),Asn-Ser-Gly-Ala-Ile-Thr-Ile-Gly (NSGAITIG), and combinations thereof atconcentrations of about 0.5% to about 10% based on the weight of thepolymer.

Coagulation Solution

The method may further comprise contacting the fibers with a coagulationsolution that comprises a coagulant to modify the water stability andmechanical properties of the fibers (e.g., tensile properties,compressive properties, abrasion properties, and bending properties).Exemplary coagulants include methanol, ethanol, sodium sulfate andacetic acid, acetone, sulfuric acid, hydrochloric acid, and combinationsthereof. In an embodiment of the present invention, the coagulant isselected from the group consisting of methanol, ethanol, sodium sulfateand acetic acid, and combinations thereof. The amount of coagulant inthe coagulant solution is such that the concentration of coagulant istypically about 1% to about 100%. Typically, the fibers are contactedwith the coagulation solution for a duration of about 5 minutes to about24 hours. Also, the coagulation solution is typically at a temperaturethat is from about 20° C. to about 100° C.

Fibers and Fibrous Structure

The above described method may be performed to make a randomly-oriented3-D fibrous structure. Such a fibrous structure comprises: one or morefibers that comprise a polymer-surfactant complex, wherein the fiber(s)have lengths that are at least about 100 nm and finenesses that areabout 50 nm to about 100 μm, and are arranged randomly and evenly inthree dimensions throughout the randomly-oriented 3-D fibrous structure;and interconnected pores having sizes that are about 10 to 2,000 μm,wherein the pores comprise about 60 to about 99.9% by volume of therandomly-oriented 3-D fibrous structure. Advantageously, experimentalresults to date indicated that such randomly-oriented,three-dimensionally fibrous structures may be used to facilitate cellsto grow and spread in three dimensions in a manner similar to that seenin native ECMs.

In another embodiment, the fibers have a fineness that is about 50 nm toabout 100 μm and the randomly-oriented 3-D fibrous structure furthercomprises interconnected pores having sizes that are about 10 to 2000 μmand has a porosity that is about 60 to about 99.9% by volume.

In yet another embodiment, the fibers have a fineness that is about 50nm to about 20 μm and the randomly-oriented 3-D fibrous structurefurther comprises interconnected pores having sizes that are about 10 to1000 μm and has a porosity that is about 90 to about 99.9% by volume ofthe structure.

In an embodiment, the one or more fibers consist of thepolymer-surfactant complex. In another embodiment, the randomly-oriented3-D fibrous structure consists of the one or more fibers and theinterconnected pores. In yet another embodiment, the randomly-oriented3-D fibrous structure consists of one or more fibers and theinterconnected pores, wherein the one or more fibers consist of thepolymer-surfactant complex.

Heat Treating the Fibers

After the fibers/fibrous structure are formed it may be heat treated ata temperature that is about 70 to 150° C. to modify the water stabilityand mechanical properties of the fibers (e.g., tensile properties,compressive properties, abrasion properties, and bending properties).

EXAMPLES General Procedures

Scaffold Preparation

Thirty 2D zein scaffolds were prepared by electrospinning 25 wt % zein(Freeman Industries LLC, Tuckahoe, N.Y.) in 70% v/v aqueous ethanol (EMDChemicals Inc., Gibbstown, N.J.) solution. Three dimensional zeinscaffolds were prepared by electrospinning aqueous solution containing25 wt % zein and 25 wt % SDS. A concentration of 9 wt % (based on theweight of zein) citric acid (EMD Chemicals Inc., Gibbstown, N.J.) wasadded into both 2D and 3D spinning dopes for cross-linking. Differentsolvent systems were utilized since zein could not be dissolved inwater. The 2D PEG scaffold was prepared by electrospinning 10 wt % PEG(50 kDa, Sigma-Aldrich, St. Louis, Mo.) aqueous solution. The 3D PEGscaffold was prepared by electrospinning 10 wt % PEG and 10 wt % SDS inaqueous solution. All the electrospinning parameters, including theextrusion speed of 2 mL/hr, voltage of 42 kV, and distance from theneedle to the collecting board of 25 cm, were kept the same for all thesamples. The needle was negatively charged, and the collecting board waspositively charged.

Morphologies and Structures of Scaffolds

The 2D and 3D scaffolds were observed using a scanning electronmicroscope (S3000N, Hitachi Inc. Schaumburg, Ill.) and a Nikon A1confocal laser scanning microscope (Nikon Inc., Melville, N.Y.).

Specific Pore Volume

Specific pore volume indicating volume of pore in unit mass of scaffoldsas shown in the following equation was selected to evaluate fluffinessof the scaffolds:

$V_{sp} = {\frac{V_{pore}}{m_{scaffold}} = {\frac{V_{scaffold}}{m_{scaffold}} - \frac{1}{\rho_{material}}}}$

where V_(sp) is the specific pore volume, V_(pore) is the volume ofpores encompassed in the scaffolds, m_(scaffold) is the mass ofscaffolds, V_(scaffold) is the volume of the scaffolds after precisemeasurement of the length, width, and thickness of scaffolds, andρ_(material) is the density of the material.

Surface Resistivity

Since the surface resistivity of ultrafine fibers is very difficult totest, films containing same polymer to surfactant/salt ratio withrelevant electrospun fibers were prepared to measure the surfaceresistivity. The films were casted onto Teflon coated plates and driedat 20° C. and 65% relative humidity. Surface resistivity was measured byemploying a surface resistivity tester (Monroe Electronics Inc.,Lyndonville, N.Y.) according to ASTM D-257 standard.

Fiber Deposition Process

A CCD camera with a longworking-distance lens was used in capturing themoment photographs of fiber deposition and scaffold formation. The timeinterval for each consequential photograph was 0.125 seconds.

Cell Attachment and Proliferation

NIH 3T3 mouse fibroblast cells (ATCC CRL-1658, Manassas, Va.) werecultured to quantitatively estimate effects of 2D and 3D structures ofzein scaffolds on cell attachment and proliferation. Cells were culturedin culture medium at 37° C. in a humidified 5% CO2 atmosphere.Electrospun 2D and 3D zein scaffolds were first rinsed in 60 wt %acetone (BDH, West Chester, Pa.) aqueous solution containing 5 wt %potassium chloride (Fisher Scientific, Fair Lawn, N.J.) to remove SDS,washed in distilled water three times, and then lyophilized. MTS assayswere performed to quantitatively investigate cell viability atattachment and proliferation stages. Samples were prepared with sameweight and then were subjected to sterilization at 120° C. for 1 hour.After sterilization, the scaffolds were placed in 48-well culture plates(TPP Techno Plastic Products, Switzerland). Fibroblast cells were seededonto the scaffolds (1×10⁵ cells mL⁻¹, 500 μL well⁻¹) and then culturedat 37° C. in a humidified 5% CO₂ atmosphere for different timeintervals. At each time point, the samples were washed with PBS, placedin new 48-well plates containing 450 μL well⁻¹ 20% MTS reagent(CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promenade)in Dulbecco's modified Eagle's medium (DMEM) and incubated at 37° C. ina humidified 5% CO₂ atmosphere for 3 hours. After incubation, 150 μL ofthe solution from each well was pipetted into a 96-well plate and theoptical densities were measured at 490 nm using a UV/vis multiplatespectrophotometer (Multiskan Spectrum, Thermo Scientific). The MTSsolution in DMEM without cells served as the blank.

Cell Penetration and Spreading

To compare penetration ability of cells on 2D and 3D scaffolds, cellswere stained by Phalloidin 633 solution (1:200 Alexa Fluor 633Phalloidin, Invitrogen, Grand Island, N.Y.) and observed using a NikonA1 confocal laser scanning microscope (Nikon Inc., Melville, N.Y.).Alexa Fluor 633 Phalloidin is a far red fluorescent dye thatspecifically bonds to F-actin in cells. This dye was selected since zeinshows fluorescence across the full spectrum with the weakest signal inthe far red range. To observe the spreading behaviors and stereoscopicmorphologies of cells in 2D and 3D scaffolds, cells were stained byPhalloidin 633 solution for F-actin and Hoechst 33342 solution(Invitrogen, Grand Island, N.Y.) for the nuclei of cells.

Statistical Analysis

One-way analysis of variance with Tukey's pairwise multiple comparisonswas employed to analyze the data. The confidence interval was set at95%, and a P value less than 0.05 was considered to be a statisticallysignificant difference. In the results, data labeled with differentsymbols were significantly different from each other.

3D Electrospun Zein Scaffolds

Ex. 1: Morphology of 3D Electrospun Zein Scaffolds

A piece of 3D zein scaffold was prepared by electrospinning aqueoussolution containing 25 wt % of zein and 25 wt % of sodium dodecylsulfate (SDS), and a piece of 2D zein scaffold was prepared byelectrospinning 25 wt % zein in 70% v/v aqueous ethanol solution; 9 wt %of citric acid based on the weight of zein was added into both 2D and 3Dspinning dopes for crosslinking. Different solvent systems were utilizedbecause the zein, itself, could not be dissolved in water. Theelectrospinning process was conducted at an extrusion speed of 2 mL/hr,a voltage of 42 kV, and a distance from the needle to the collectingboard of 25 cm. The needle was negatively charged and the collectingboard was positively charged.

As is shown in FIG. 3, the 3D electrospun zein scaffold hassignificantly higher porosity and fluffiness than its 2D counterpartwith the same weight. FIG. 4 contains the microscopic morphologies of 3Delectrospun zein scaffolds observed under scanning electron microscope(SEM, left) and confocal laser scanning microscope (CLSM, right), whichshow that the orientation of fibers in the 3D electrospun zein scaffoldwas random (from both the top surface and the side).

Ex. 2: In Vitro Cell Culture Study of 3D and 2D Electrospun ZeinScaffolds

NIH 3T3 fibroblast cells were cultured on both 2D and 3D electrospunzein scaffolds to evaluate the effects of scaffold architecture oncellular attachment, penetration and proliferation. Cell penetration wasevaluated 48 hours after culturing the 2D and 3D scaffolds.Significantly higher cell accessibility of 3D zein electrospun scaffoldcompared with 2D scaffold is shown in FIG. 5. Cells cultured on the 3Delectrospun zein scaffold were found at the depth of 120 μm from thesurface, whereas cells could not be seen 40 μm below the surface of the2D electrospun zein scaffold. It is believed that the “looser” structureof the 3D electrospun scaffold accounted for the much better cellularpenetration in the 3D scaffold.

Methanethiosulfonate (MTS) assays were conducted to quantitativelyinvestigate cell attachment and proliferation, the results of which areshown in FIG. 6. The amount of cells attached on the 3D zein scaffold 4hours after seeding was 114% higher than that on the 2D scaffold. Aremarkably higher proliferation rate was also found for cells cultivatedon 3D zein scaffold. Cell proliferation on the 3D scaffold reachedplateau 5 days after seeding and the cell density increased more than 4times, while that on 2D scaffold reached plateau 3 days after seedingand the increase in cell density was less than twice. It is believedthat the greater cyto-accessible space of the 3D scaffold may be thereason for higher cell accessibility and better proliferation.

3D zein scaffolds showed a great potential for tissue engineeringapplications. For example, FIG. 7 b shows spheroid-shaped cells on 3Dscaffolds whereas FIG. 7 a shows cells with flattened morphologies on 2Dscaffolds. More specifically, FIG. 7 a.I shows he developedcytoskeletons of cells over the surface of 2D scaffold (the x-yprojection). The thickness of the cells illustrated in the x-zprojection (FIG. 7 a.II) and the y-z projection (FIG. 7 a.III) was muchsmaller than their planar sizes in the x-y projection (FIG. 7 a.I). Ascan be seen, cells seeded on the 2D scaffolds developed into 2D planarmorphologies. In contrast, the side views of 3D scaffolds in FIG. 7b.II′ and III′ show that the cells oriented in thickness directionrather than in planar x and y directions. It is believed that the 3Dscaffolds facilitated the cells to develop into stereoscopicarchitectures that more closely mimic the cells in many native ECMs.

3D Electrospun PEG Scaffolds Using Anionic and Nonionic Surfactants

Ex. 3: Morphology of 3D Electrospun PEG Scaffolds Using AnionicSurfactant

PEG is a water soluble synthetic polymer, and could also be electrospuninto 3D stereoscopic architecture. PEG (25 wt %) and SDS (25 wt %) weredissolved in water to prepare spinning dopes. The electrospinningprocess was conducted at an extrusion speed of 2 mL/hr, a voltage of 42kV, and a distance from the needle to the collecting board of 25 cm. Theneedle was negatively charged and the collecting board was positivelycharged. As is shown in FIG. 8, the 3D electrospun zein scaffold hassignificantly higher porosity and fluffiness than its 2D counterpartwith the same weight. The 3D PEG electrospun scaffold was composed ofspatially randomly oriented fibers with diameter around 0.8 μm.

Ex. 4: Morphology of 3D Electrospun PEG Scaffolds Using NonionicSurfactant

PEG (25 wt %) and TRITON X-100 (25 wt %), a nonionic surfactant, wasdissolved in water for spinning. The electrospinning process wasconducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and adistance from the needle to the collecting board of 25 cm. The needlewas negatively charged and the collecting board was positively charged.As is shown in FIG. 9, the 3D electrospun zein scaffold hassignificantly higher porosity and fluffiness than its 2D counterpartwith the same weight. The 3D PEG electrospun scaffold was composed ofspatially randomly oriented fibers with diameter around 0.8 μm.

3D Electrospun Scaffold From Water Insoluble Highly Crosslinked Proteins

Ex. 5: Extraction of Spinable Proteins from Soy Protein and WheatGlutenin

Soy protein and wheat glutenin represent two types of highly crosslinkedproteins that are high in molecular weights and insoluble in water.These proteins were treated in 8 M urea under mild alkaline conditionwith existence of cysteine, a common amino acid, which is also anontoxic and environmentally benign reductant. After being treated at70° C. for 24 hours, the soluble proteins were collected. SDS PAGEresults of these extracted proteins showing molecular weights are shownin FIG. 10. Lane 1 showed standard protein markers. Lanes 2, 3, and 4were untreated soy protein, cysteine extracted soy protein and soyprotein treated with NaOH. The major bands (20 kDa, 37 kDa and 63 kDa)of soy protein remained in the extracted sample, while no obvious bandscould be found in the NaOH treated sample. Lane 5 and 6 were wheatglutenin and cysteine extracted wheat glutenin. The highly similar bandsof the two samples revealed the high efficient yet non-destructivemanner of cysteine extraction.

Ex. 6: Morphology of 3D Electrospun Scaffold from Soy Protein

Soy protein extracted using protocol as mentioned in Example 4 (25 wt %)and 25 wt % of SDS were dissolved in water and electrospun into 3Dstructures as shown in FIG. 11. The electrospinning process wasconducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and adistance from the needle to the collecting board of 25 cm. The needlewas negatively charged and the collecting board was positively charged.The diameter of the ultrafine fibers ranged from 1 to 3 μm.

Ex. 7: Water Stability of 3D Electrospun Soy Protein Scaffold

After post treatment of coagulation bath of 10% Na₂SO₄ and 10% aceticacid, the 3D electrospun soy protein scaffold was soaked in PBS at 50°C. for 3 days. As shown in FIG. 12, the scaffold retained fibers intheir structures as the diameters of fibers increased from 1-3 μm to 2-5μm after 3 days. The 3D electrospun soy protein scaffold showed fairlygood water stability.

Ex. 8 Morphology of 3D Electrospun Scaffold from Feather Keratin

Feather keratin extracted using protocol as mentioned in Example 4 (25wt %), 25 wt % of SDS and cysteine (10 wt % based on keratin) weredissolved in water and electrospun into 3D structures as shown in FIG.13. The electrospinning process was conducted at an extrusion speed of 2mL/hr, a voltage of 42 kV, and a distance from the needle to thecollecting board of 25 cm. The needle was negatively charged and thecollecting board was positively charged. The diameter of the ultrafinefibers ranged from 2 to 5 μm.

Ex. 9 Morphology of 3D Electrospun Scaffold from Wheat Glutenin

Wheat glutenin extracted using protocol as mentioned in Example 4 (25 wt%), 25 wt % of SDS and cysteine (10 wt % based on wheat glutenin) weredissolved in water and electrospun into 3D structures as shown in FIG.14. The electrospinning process was conducted at an extrusion speed of 2mL/hr, a voltage of 42 kV, and a distance from the needle to thecollecting board of 25 cm. The needle was negatively charged and thecollecting board was positively charged. The diameter of the ultrafinefibers around 2 μm.

3D Electrospun Scaffold From Other Proteins

Ex. 10: Morphology of 3D Electrospun Scaffold from Wheat Gliadin

Wheat gliadin (25 wt %), a prolamin in wheat, and 25 wt % of SDS weredissolved in water and electrospun into 3D structures as shown in FIG.15. The electrospinning process was conducted at an extrusion speed of 2mL/hr, a voltage of 42 kV, and a distance from the needle to thecollecting board of 25 cm. The needle was negatively charged and thecollecting board was positively charged. The diameter of fibers in 3Dgliadin electrospun scaffold ranged from 1.5 to 3.5 μm.

Ex. 11: Morphology of 3D Electrospun Scaffold from Casein

Casein (25 wt %), the family of proteins commonly found in mammalianmilk, and 25 wt % of SDS were dissolved in water and electrospun into 3Dstructures as shown in FIG. 16. The electrospinning process wasconducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and adistance from the needle to the collecting board of 25 cm. The needlewas negatively charged and the collecting board was positively charged.The diameter of fibers in 3D casein electrospun scaffold ranged from 0.8to 2.8 μm.

Ex. 12: Morphology of 3D Electrospun Scaffold from Peanut Protein

Peanut protein (25 wt %) and 25 wt % of SDS were dissolved in water andelectrospun into 3D structures as shown in FIG. 17. The electrospinningprocess was conducted at an extrusion speed of 2 mL/hr, a voltage of 42kV, and a distance from the needle to the collecting board of 25 cm. Theneedle was negatively charged and the collecting board was positivelycharged. The diameter of fibers in 3D peanut protein electrospunscaffold ranged from 0.9 to 2.9 μm.

Relationship between Specific Pore Volume and Surface Resistivity

A power function was used to simulate the relationship between thespecific pore volume of electrospun scaffolds and corresponding polymersurface resistivity. As shown in FIG. 18 a, the residual standard errorof the model is 0.613, which is a reasonable number to indicate that thedata could be well described by power function and suggests that thereis a strong quantitative relationship between the specific pore volumeand surface resistivity. The power function was constructed as y=ax^(b)where y represented specific pore volume, x represented surfaceresistivity, and a and b were coefficients varied with the type ofmaterials. Here, for PEG, a equaled to 2.208×105 and b equaled to−0.5325. The specific pore volume decreased exponentially as surfaceresistivity increased. As surface resistivity decreased from 109 to 106Ω/sq., the macrostructure of PEG scaffolds converted from 3D to 2D, andthe specific pore volume decreased by about 20 times. It may be inferredthat the increased surface conductivity increased fluffiness ofscaffolds exponentially. It was found that surface resistivity of thepolymer decreased with increasing SDS proportion. As shown in FIG. 18 b,surface resistivity of PEG decreased as SDS content increased. Surfaceresistivity of pure PEG was higher than 109 Ω/sq. When the weight ratioof SDS to PEG was increased to 1:1, surface resistivity was reducedconsiderably to 106 Ω/sq. However, when NaCl was added into the polymer,the surface resistivity did not decrease as substantially as the samemole of SDS was added. This is because when water evaporated, SDS mainlydistributed on the surface of polymer while NaCl may distribute moreevenly in the polymer. The sulfate groups of SDS that concentrated onthe surface of fiber oriented toward the outside and could induceformation of a surface water layer on the fibers. In the surface waterlayer of PEG fibers, free movement of dissociable sodium ions from SDSeffectively decreased surface resistivity of PEG electrospun fibers.Whereas the evenly distributed NaCl would only decrease the volumeresistivity but could not effectively decrease surface resistivity offibers. In summary, by adding SDS, the polymer was converted frominsulator to semiconductor, the capability of transferring staticelectricity of the fiber has been tremendously increased, and thiscorrespondingly increased the fluffiness of scaffolds.

To further investigate the effect of electron transference on formationof 3D architectures, a solution with 25 wt % zein and 25 wt % SDS waselectrospun onto the positively charged collecting board covered by alayer of insulator. Delivery of electrons was interrupted thoughpositive potential still existed. Zein fibers with electrons on thesurface were attracted by the positive collector and then hit the boardvertically as shown in FIG. 19 a. However, the electrons could not betransferred onto the collecting board and thus remained on the fibers.The highly negatively charged fibers attached onto the insulatortightly, owing to the strong electrical attraction, and consequently atraditional 2D electrospinning scaffold (FIGS. 19 b and c) was formed.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles.

Although the materials and methods of this invention have been describedin terms of various embodiments and illustrative examples, it will beapparent to those of skill in the art that variations can be applied tothe materials and methods described herein without departing from theconcept, spirit and scope of the invention. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A method of making a randomly-oriented 3-Dfibrous structure, the method comprising electrospinning a spinning dopewith an electrospinning apparatus, wherein the spinning dope comprises:a solvent; a polymer dissolved in the solvent, wherein the dissolvedpolymer is in subunits having molecular weights that are about 5 toabout 150 kDa; and a surfactant; to form one or more fibers thatcomprise a polymer-surfactant complex and that arrange randomly andevenly in three dimensions when contacting a collecting board of theelectrospinning apparatus thereby forming the randomly-oriented 3-Dfibrous structure.
 2. The method of claim 1, wherein the polymer isselected from the group consisting of protein, synthetic polymer, andcombinations thereof.
 3. The method of claim of claim 2, wherein theprotein is selected from the group consisting of plant protein, animalprotein, and combinations thereof.
 4. The method of claim 1, wherein thespinning dope has a concentration of the surfactant that is about 5 toabout 300 percent by weight of the polymer.
 5. The method of claim 1,wherein the surfactant is selected from the group consisting of anionicsurfactant, cationic surfactant, nonionic surfactant, zwitterionicsurfactant, and combinations thereof.
 6. The method of claim 1, whereinthe solvent is selected from the group consisting of water, phosphatebuffered saline (PBS), carbonate buffer, tris-glycine buffer, boratebuffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid (CHES)buffer, citric buffer, ethanol, chloroform, 1,4-dioxane, methanol,ethylene glycol, acetone, ethyl acetate, methyl acetate, hexane, petrolether, citrus terpenes, diethyl ether, dichloromethane,dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO),formic acid, n-butanol, isopropanol (IPA), n-propanol, acetic acid,nitromethane, dichloromethane, and combinations thereof.
 7. The methodof claim 1, wherein: the fibers have a fineness that is about 50 nm toabout 100 μm; the randomly-oriented 3-D fibrous structure furthercomprises interconnected pores having sizes that are about 10 to 2000μm; and the randomly-oriented 3-D fibrous structure has a porosity thatis about 60 to about 99.9% by volume.
 8. The method of claim 1, wherein:the polymer consists of a primary polymer and one or more secondarypolymers and the spinning dope further comprises a first cross-linker, asacrificial component, or a combination thereof; or the method furthercomprises contacting the fibers with a second solution that comprises asecond cross-linker, a surface decorator, or a combination thereof; or acombination thereof.
 9. The method of claim 8, wherein the firstcross-linker is at a concentration of about 0.01 mol/L to about 10 mol/Lof the spinning dope, and wherein the sacrificial component is at aconcentration of about 0.5% to about 50% based on the weight of thepolymer, and wherein the second solution has a concentration of thesecond cross-linker that is about 0.01 mol/L to about 10 mol/L, andwherein the surface decorator is at concentration of about 0.5% to about10% based on the weight of the polymer.
 10. The method of claim 9,wherein the first and second cross-linkers are independently selectedfrom the group consisting of polycarboxylic acid which contains at leastthree carboxylic acid groups, oxysucrose, genepin, glutaraldehyde,oxaldehyde, NHS esters, maleimides, carbodiimide, isocyanate, andcombinations thereof, and wherein the sacrificial component is selectedfrom the group consisting of PEG, egg white protein, zein, wheatgliadin, and combinations thereof, and wherein the surface decorator isone or more peptides selected from the group consisting of Arg-Gly-Asp(RGD), Ile-Lys-Val-Ala-Val (IKVAV), and Asn-Ser-Gly-Ala-Ile-Thr-Ile-Gly(NSGAITIG).
 11. The method of claim 1, wherein the polymer is a proteinand the method further comprises aging the dissolved protein at an agingtemperature that is about 20 to about 90° C. for an aging duration thatis about 0.5 to about 48 hours before conducting the electrospinning.12. The method of claim 1, further comprising contacting the fibers witha coagulation solution that comprises a coagulant to modify the waterstability and mechanical properties of the fibers.
 13. The method ofclaim 12, wherein the coagulant is selected from the group consisting ofmethanol, ethanol, sodium sulfate and acetic acid, acetone, sulfuricacid, hydrochloric acid, and combinations thereof.
 14. A method ofmaking a randomly-oriented 3-D fibrous structure, the method comprisingelectrospinning a spinning dope with an electrospinning apparatus,wherein the spinning dope comprises: a solvent; a polymer dissolved inthe solvent, wherein the dissolved polymer is in subunits havingmolecular weights that are about 10 to about 50 kDa, and wherein thepolymer is selected from the group consisting of protein, syntheticpolymer, and combinations thereof; and an anionic surfactant at about 5to about 300 percent by weight of the polymer; to form one or morefibers of a fineness that is about 50 nm to about 100 μm and thatcomprise a polymer-surfactant complex and that arrange randomly andevenly in three dimensions when contacting a collecting board of theelectrospinning apparatus thereby forming the randomly-oriented 3-Dfibrous structure that further comprises interconnected pores havingsizes that are about 10 to 2000 μm and that has a porosity that is about60 to about 99.9% by volume.
 15. A randomly-oriented 3-D fibrousstructure comprising: one or more fibers that comprise apolymer-surfactant complex, wherein the fiber(s) have lengths that areat least about 100 nm and finenesses that are about 50 nm to about 100μm, and are arranged randomly and evenly in three dimensions throughoutthe randomly-oriented 3-D fibrous structure; and interconnected poreshaving sizes that are about 10 to 2,000 μm, wherein the pores compriseabout 60 to about 99.9% by volume of the randomly-oriented 3-D fibrousstructure.
 16. The randomly-oriented 3-D fibrous structure of claim 15,wherein: the fineness of the one or more fibers is about 50 nm to about20 μm; and the interconnected pores have sizes that are about 100 to1,000 μm, wherein the pores comprise about 90 to about 99.9% by volumeof the randomly-oriented 3-D fibrous structure.
 17. Therandomly-oriented 3-D fibrous structure of claim 15, wherein thepolymer-surfactant complex is formed via electrospinning of a spinningdope that comprises a solvent; a polymer dissolved in the solvent,wherein the dissolved polymer is in subunits having molecular weightsthat are about 5 to about 150 kDa; and a surfactant.
 18. Therandomly-oriented 3-D fibrous structure of claim 17, wherein thesurfactant is selected from the group consisting of anionic surfactant,cationic surfactant, nonionic surfactant, zwitterionic surfactant, andcombinations thereof; and wherein the polymer is selected from the groupconsisting of protein, synthetic polymer, and combinations thereof. 19.The randomly-oriented 3-D fibrous structure of claim 18, wherein theprotein is selected from the group consisting of plant protein, animalprotein, and combinations thereof.
 20. The randomly-oriented 3-D fibrousstructure of claim 19, wherein: the plant protein is selected from thegroup consisting of wheat gluten, wheat gliadin, wheat glutenin, soyprotein, camelina protein, peanut protein, canola protein, sorghumprotein, rice protein, millet protein, sunflower seed protein, pumpkinseed protein, mung bean protein, red bean protein, chickpea protein,green pea protein, and combinations thereof; the animal protein isselected from chicken feather, egg white, wool keratin, casein, silk,fibrin, collagen, gelatin, hair keratin, horn keratin, nail keratin,whey protein, and combinations thereof; and the synthetic polymer isselected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), poly glycolic acid (PGA), polyhydroxyalkanoates(PHAs), poly(lactic-co-glycolic acid) (PLGA), poly-3-hydroxybutyrate(PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH),poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and combinationsthereof.